Zhu Fm

Molecular basis for an individual with rare p phenotype in P1Pk blood group system

OBJECTIVE To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system. METHODS Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing. RESULTS The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position. CONCLUSION The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.

[Establishment of a polymerase chain reaction sequencing based typing method for HLA-DPB1 exons 2 and 3 and investigation of their polymorphisms].

OBJECTIVE To establish a polymerase chain reaction sequencing-based typing (PCR SBT) method for HLA-DPB1 exons 2 and 3, and to analyze their polymorphisms. METHODS Based on the sequences of HLA-DPB1 loci, locus-specific primers were designed and applied to amplify the target sequences encompassing the entire exons 2 and 3 of HLA-DPB1. The amplification products were digested by enzymes and directly sequenced in both directions. The genotype was assigned by Assign 3.5+ SBT software. RESULTS Specific target fragment was obtained with the PCR amplification, and good quality electropherogram was derived by direct sequencing. Among 242 individuals from Zhejiang Han population, 18 HLA-DPB1 alleles were detected. Alleles with a frequency of > 0.05 have included DPB1*05:01:01/135:01 (0.4112), DPB1*02:01:02 (0.1901), DPB1*04:01:01 (0.1136) and DPB1*02:02 (0.0620). A novel HLA-DPB1*168:01 allele has also been identified. Nine polymorphism sites were founded in the exon 3 region, which included a new SNP site 517 A>T. CONCLUSION The PCR-SBT method for exons 2 and 3 of HLA-DPB1 is reliable, which allowed detection of polymorphisms in exon 3 of the HLA-DPB1 gene.

[Analysis of allele frequencies of HLA-DRB1*12:01:01G and HLA-DRB1*14:01:01G groups].

OBJECTIVE To discriminate and analyze the relative frequencies of alleles in HLA-DRB1*12:01:01G(HLA-DRB1*12:01:01/12:06/12:10/12:17) and HLA-DRB1*14:01:01G (DRB1*14:01:01/14:54) groups and assess their associations with HLA-DRB3 and HLA-DQB1 loci. METHODS A total of 115 DNA samples previously typed as HLA-DRB1*12:01:01G and 108 samples from HLA-DRB1*14:01:01G were selected. DNA sequences for exons 1 to 3 of the HLA-DRB1 locus were analyzed for HLA-DRB1*12:01:01G, and exons 2 to 3 were analyzed for HLA-DRB1*14:01:01G by polymerase chain reaction sequence-based typing (PCR-SBT). Genotyping of HLA-DRB3 and HLA-DQB1 were achieved by PCR-SBT. RESULTS Among 115 samples previously typed as HLA-DRB1*12:01:01G, 101 (87.8%) were confirmed as HLA-DRB1*12:01:01 and 14 (12.2%) were HLA-DRB1*12:10, but HLA-DRB1*12:06 and HLA-DRB1*12:17 alleles were not identified. For 108 samples previously typed as HLA-DRB1*14:01:01G, all were typed as HLA-DRB1*14:54. HLA-DRB1*12:01:01 was linked with HLA-DRB3*01:01:02 and HLA-DQB1*03:01, while HLA-DRB1*12:10 was strongly linked with HLA-DRB3*02:02:01 and HLA-DQB1*03:01. HLA-DRB1*14:54 was strongly linked with HLA-DRB3*02:02:01 and two different HLA-DQB1*05:02, *05:03 alleles. CONCLUSION HLA-DRB1*12:01:01 was more prevalent than HLA-DRB1*12:10 in the HLA-DRB1*12:01:01G group, and HLA-DRB1*14:54 was the dominant allele for HLA-DRB1*14:01:01G.

[Study of in vitro expression of human platelet ITGB3 gene nonsense mutation c.1476G>A].

OBJECTIVE To explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system. METHODS An eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively. RESULTS The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis. CONCLUSION The ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

[A rare Pk phenotype caused by a 433 C>T mutation of the β-1,3-N-acetylgalactosyltransferase gene].

OBJECTIVE To study the serological characteristics and molecular mechanism for a rare Pk phenotype of the P1Pk blood group system. METHODS The blood group of the proband was identified by serological techniques. The coding region and flanking intronic sequences of the β-1,3-N-acetylgalactosyltransferase gene (B3GALANT1) associated with the Pk phenotype were analyzed using polymerase chain reaction sequence-based typing. RESULTS The proband was identified as having a rare Pk phenotype including anti-P in her serum. The blood group of her daughter and husband showed a P2 phenotype. The nucleotide sequences of the B3GALANT1 gene of her husband and two randomly-chosen individuals were the same as the reference sequence (GenBank AB050855). Nucleotide position 433 C>T homozygous mutation in the B3GALANT1 was found in the proband, which has resulted in a stop codon at amino acid position 145, which may produce a premature protein capable of decreasing or inhibiting the activity of the β -1,3-N-acetylgalactosyltransferase. The nucleotide position 433 C/T heterozygous in the B3GALANT1 was found in her daughter. CONCLUSION The Pk phenotype resulted from 433 C>T mutation in the B3GALANT1 gene has been identified.

[Development of a method for the separation of HLA-A, -B and -C haploid using biotinylated probe and streptavidin magnetic beads].

OBJECTIVE To develop a method for separating the human leukocyte antigen (HLA)-A, -B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results. METHODS Based on sequence information of HLA alleles from the IMGT/HLA database, the 5-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis. RESULTS Among the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples. CONCLUSION The developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.

[Analysis of erythroid-specific blood group genes using un-mobilized peripheral stem cells cultured in vitro].

OBJECTIVE To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro. METHODS Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting, followed by suspension culture in vitro. Cells were collected from medium on various stages and analyzed by immunofluorescence. The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12. RESULTS A total of(3.19±0.13) ×10 (4) CD34+cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3%±2.7%. The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input. The stem cell-specific CD34 antigen was dropped off, while the erythroid-specific CD235a and CD240D antigens were increased in culture period. RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12, and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes. CONCLUSION A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.

Mutation analysis and prenatal diagnosis of keratin 9 gene in a large Chinese family with epidermolytic palmoplantar keratoderma

OBJECTIVE To analyze potential mutation in keration 9 (KRT9) gene in a large Chinese family with epidermolytic palmoplantar keratoderma (EPPK) and to perform prenatal diagnosis on the fetus at 10th gestational week. METHODS Peripheral venous blood samples were obtained from 5 affected and 8 unaffected individuals of the family. Fifty unrelated healthy individuals were also recruited as controls. PCR was used to amplify exons 1 and 6 of KRT9 gene, and the products were sequenced directly. After the mutation was confirmed, prenatal diagnosis was performed on the fetus during the first trimester of pregnancy. RESULTS A heterozygous missense mutation c.482A to G in the KRT9 gene, which has led to substitution of Asparaginate by Serine at codon 161 (p.N161S), was detected in all patients but not in other individuals of the family and the 50 healthy controls. The fetus was found to have carried the p.N161S mutation too. Following selected abortion, analysis of fetal tissue was consistent with prenatal diagnosis. CONCLUSION The missense mutation c.482A to G (p.N161S), which has been shown previously to cause EPPK, is found in the KRT9 gene of patients in this family. Gene mutation analysis for prenatal diagnosis is efficient to facilitate detection of affected fetus in time.

[A rare p phenotype caused by a 26-bp deletion in α 1,4-galactosyltransferase gene].

OBJECTIVE To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual. METHODS Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing. RESULTS A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the probands family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted. CONCLUSION A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.

Study on a novel mutation of B glycosyltransferase gene related with an ABx variant

OBJECTIVE To explore the molecular basis of an individual featuring an ABx variant of ABO blood group system. METHODS Serological assays were used to characterize the erythrocyte phenotypes and salivary ABH secretors. All of the seven exons and flanking introns of ABO glycosyltransferase gene were amplified with polymerase chain reaction (PCR). And the products were sequenced bidirectionally following enzyme digestion. Exons 6 and 7 were also subcloned and analyzed for haplotypes of the ABO gene. RESULTS Erythrocytes of the proband have expressed a strong A antigen and a weak B antigen, which was identified as a rare ABx variant in addition with other serological features. Nine heterozygous sites in exon 6 (297A/G) and exon 7 (467C/T, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 808T/A, 930G/A) of the coding region of the ABO gene were identified. Based on haplotype analysis, one allele was determined as common A102, whilst another was consistent with B101 except for an 808T>A mutation which has resulted in replacement of phenylalanine with isoleucine at position 270 of glycosyltransferase B. CONCLUSION The 808T>A mutation of the glycosyltransferase B gene may decrease the enzymatic activity and result in the Bx variant.